This invention is generally in the area of ophthalmic compositions and more specifically relates to a method and compositions for enhancing healing of injuries to the corneal endothelium.
The corneal endothelium, a single layer of hexagonal cells at the boundary between the fluid-filled anterior chamber and the clear collagenous stroma at the posterior surface of the cornea, is critical for the maintenance of transparency of the tissue. The cell layer is avascular, lacks innervation, and is bathed on its apical face by aqueous humor. In the aging human, the density of corneal endothelial cells gradually decreases and the remaining cells retain their polygonal shape while becoming wider and thinner to maintain a permeability barrier. When a wound or discontinuity occurs in this tissue, the cells bordering the defect become elongated, migratory, and, perhaps, synthesize new extracellular matrix material.
Clinically significant dysfunctions of the endothelium, due to cell loss associated with dystrophies or degenerations caused by ocular diseases, drugs, trauma or surgery, are responsible for the majority of corneal transplants. Despite its physiologic importance, the corneal endothelium is an extremely fragile tissue. Since the endothelium is relatively amitotic in the adult human, repair to areas of injury is via migration and spreading of neighboring cells to cover the defect in the monolayer. At low cell densities, these repair mechanisms may be insufficient to restore the endothelium to full physiologic function. If the endothelial barrier and pump functions become compromised, water enters the stroma and disrupts the precise arrangement of collagen fibrils, eventually resulting in visual impairment.
Upon disruption of the endothelial barrier, undamaged cells break their junctional complexes, elongate and migrate to fill denuded areas. These areas of repair are characterized by stromal edema. Following repopulation of the area, cells reform their junctions, resume their hexagonal shape, and regain their ability to act as a permeability barrier, with subsequent resolution of corneal edema.
The corneal endothelium has been characterized biochemically, physiologically and ultrastructurally. Features include abundant NA/K ATPase, adenylate cyclase activity, cyclooxygenase/lipooxygenase pathways, EGF and .beta.-adrenergic receptors, phenotypically controlled synthesis of type IV collagen, active fluid secretion coupled to bicarbonate transport, gap junctions, maculae occludens and highly organized circumferential bundles of f-actin.
Currently, medical management of endothelial cell loss and dysfunction is limited, and research aimed at developing drug treatments is impeded by the lack of suitable experimental models for human tissue.
Studies of the events of wound closure in tissue culture systems, even using rabbit cells which, unlike human cells, are mitotically active, allow independent observation of changes in cell shape, migration, metabolism, and proliferation. The rabbit corneal endothelium is not an ideal model because of its relatively high mitotic rate compared to human tissue. Nevertheless, the use of rabbits to study wound closure of the corneal endothelium has led to significant observations on cellular shape changes during migration as well as changes in tissue permeability and pump function during healing. Furthermore, comparing the mitotic abilities of rabbit and human endothelium may provide insight into the amitotic state of human tissue.
Studies using cultured rabbit corneal endothelial tissue were reported in Invest. Opthalmol. Vis. Sci. 25, 1235 (1984) by Neufeld, et al, Invest. Opthalmol. Vis. Sci. 27(10), 1437-1442 (October 1986) by Neufeld, et al, and Invest. Opthalmol. Vis. Sci. 27(4), 474-479 (April 1986) by Raymond, et al. The studies reported in the 1984 paper demonstrated that cultured rabbit endothelial cells, like the native tissue, synthesizes cyclic AMP in response to .beta.-adrenergic stimulation. The mitogenic and morphological responses of cultured rabbit tissue to epidermal growth factor (EGF), a well-defined growth factor which stimulates bovine, feline, and perhaps human corneal endothelial proliferation, were studied and reported in the April 1986 paper. EGF was found to induce a shape change in the cells that is independent of mitosis. It was postulated that the shape changes were similar to that found in migrating cells closing a wound. EGF-induced elongation was reversible, independent of EGF-stimulated mitosis, and similar to the elongation of migratory cells adjacent to experimental endothelial injuries. It was hypothesized that EGF exerts its effect via a specific receptor and can increase phosphotidyl inositol turnover and subsequent synthesis of arachidonic acid metabolites.
These studies indicated that epidermal growth factor (EGF) and/or indomethacin (INDO) cause elongation in first passage cultures of rabbit and human corneal endothelial cells and that addition of exogenous prostaglandin E.sub.2 (pgE.sub.2) to cultures treated with EGF and indomethacin blocks elongation, i.e., the cells retain their polygonal shape. As cultures of polygonal endothelial cells synthesize abundant pgE.sub.2 and this synthesis is totally inhibited by indomethacin, it was concluded that endogenous synthesis of pgE.sub.2 is necessary for maintenance of normal endothelial cell shape.
Epidermal growth factor (EGF) is an acidic, low molecular weight protein originally purified from mouse submaxillary gland which was initially identified because of its ectodermal effects on new-born mice, causing premature eyelid opening and incisor eruption. EGF is a 53-residue polypeptide of 6,045 Daltons, with three cysteine-cysteine linkages. Three amino acids are completely absent from its structure: lysine, alanine, and phenylalanine. EGF has been shown to stimulate proliferation of corneal epithelium in organ culture and in vivo. Recently, EGF has also been shown to stimulate proliferation of cultured corneal endothelial cells in organ culture.
While these findings are interesting, they do not provide a method for controlling and enhancing repair and restoration of normal functioning of the corneal endothelium in the human eye.
It is therefore an object of the present invention to provide methods for enhancing and controlling repair and restoration of function of the human corneal endothelium.
It is a further object of the present invention to provide compositions for the manipulation of the healing process and restoration of the corneal endothelium's function as a permeability barrier.